1. Field
This disclosure is concerned generally with a novel influenza virus propagation medium and specifically with the use of that medium for influenza vaccine production.
2. Prior Art
Influenza vaccines have been in use since the early 1940's for human vaccination and since the late 1960's for equine vaccination. All influenza vaccines presently used are made by growing the vaccine virus strains in embryonated chicken eggs. The resulting virus strains are then used for making live virus vaccines or further processed to make killed virus vaccines.
It is generally known by virologists that influenza viruses grow to a very limited degree in cell cultures. The growth is referred to as a "one-step growth cycle"; that is, only the originally infected cells replicate viruses. This phenomenon is described, for example, by Davis et al., MICROBIOLOGY, Harper and Row Publishers, Chapter 44, pp. 1138-39 (1968). Since the viruses of the originally infected cells are unable to infect successive numbers of cells in the same cell culture, the resulting yields are far too low to be useful in the preparation of virus vaccines. Thus, liquid cell cultures have not been used for commercial production of influenza virus vaccines.
Embryonated chicken eggs are used to produce viruses with titers sufficiently high enough to be useful in the preparation of vaccines. Unfortunately, chick embryo-grown viruses usually require concentration, and, in the case of human vaccines, also require some form of purification to reduce toxic reactions due to the undesirable egg proteins. The use of the eggs for vaccine production is time consuming, labor intensive, requires relatively high material costs, and the yield from one egg is commonly only enough to produce vaccine for about one to 1.5 doses. Thus, the manufacture of millions of doses requires innoculating and harvesting millions of embryonated eggs.
Recently, it has been noted that a wide variety of influenza A viruses comprising human, equine, porcine, and avian strains, grew productively in an established line of canine kidney cells under an overlay medium containing trypsin and formed well-defined plaques regardless of their prior passage history. See the article by K. Tobita et al., "Plaque Assay and Primary Isolation of Influenza A Viruses in an Established Line of Canine Kidney Cells (MDCK) in the presence of Trypsin", Med. Microbiol. Immunol. 162, 9-14 (1975). See also the article by Hans-Dieter Klenk et al., "Activation of Influenza A Viruses by Trypsin Treatment", Virology 68, 426-439 (1975). It should be noted that in the above reports the effects of trypsin on influenza virus propagation were observed in semi-solid cultures in plaque formation assays and isolation techniques, neither of which are concerned with liquid cell cultures or the large scale or commercial propagation of influenza viruses for vaccine production. The term liquid cell culture used herein describes the in vitro growth of cells and propagation of virus in a chemically defined liquid medium.
Quite surprisingly, we have now found that proteolytic enzymes can also be used in liquid cell cultures to facilitate infection of successive numbers of cells in the same cell culture. By thus overcoming the limitations of the "one-step growth cycle" of past liquid cell culture techniques, it is possible to achieve an influenza virus yield which is in the range of about 1,000 to 10,000 fold greater than non-protease treated cultures. This makes feasible the use of liquid cell culturing techniques for the commercial production of influenza vaccines, thereby avoiding the disadvantages associated with using embryonated chicken eggs. Details of our culturing medium, virus propagation techniques, and vaccine production and use methods are disclosed herein.